IDH1 mutation produces R-2-hydroxyglutarate (R-2HG) and induces mir-182-5p expression to regulate cell cycle and tumor formation in glioma

Background Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), are present in most gliomas. IDH1 mutation is an important prognostic marker in glioma. However, its regulatory mechanism in glioma remains incompletely understood. Results miR-182-5p expression was increased within IDH1-mutant glioma specimens according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples. (R)-2-hydroxyglutarate ((R)-2HG) treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p inhibition partially eliminated the oncogenic effects of R-2HG upon glioma cells. By direct binding to Cyclin Dependent Kinase Inhibitor 2 C (CDKN2C) 3’UTR, miR-182-5p inhibited CDKN2C expression. Regarding cellular functions, CDKN2C knockdown promoted R-2HG-treated glioma cell viability, suppressed apoptosis, and relieved cell cycle arrest. Furthermore, CDKN2C knockdown partially attenuated the effects of miR-182-5p inhibition on cell phenotypes. Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition in cell cycle check point and apoptosis markers. The engineered CS-NPs (antagomir-182-5p) effectively encapsulated and delivered antagomir-182-5p, enhancing anti-tumor efficacy in vivo, indicating the therapeutic potential of CS-NPs(antagomir-182-5p) in targeting the miR-182-5p/CDKN2C axis against R-2HG-driven oncogenesis in mice models. Conclusions These insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/CDKN2C axis, offering a promising therapeutic avenue against R-2HG’s oncogenic influence to glioma. Supplementary Information The online version contains supplementary material available at 10.1186/s40659-024-00512-2.


Introduction
Mutations in tricarboxylic acid cycle (TCA cycle) enzymes, such as isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), are present in several cancers, particularly gliomas [1,2].Heterozygous mutations in IDH1 and IDH2, especially those in IDH1 have been discovered within 70-80% of cases of WHO grade II and III astrocytoma, oligodendrogliomas, and secondary glioblastomas [3][4][5].Nowadays, mutated IDH1 even defines a different molecular subtype of diffuse glioma [1,6].Therefore, investigating the role and mechanism of IDH1 mutation in glioma pathogenesis could pave the way for the development of targeted therapeutic strategies that specifically address the effects of IDH1 mutations in gliomas.
The effects of IDH1 mutation upon gliomas are complex and seemingly paradoxical.Glioblastoma patients carrying an IDH1 mutation show better prognosis; however, the median overall survival remains about 31 months for gliomas patients [1] and 44 months for glioblastoma multiforme (GBM) patients [6].Notably, a mutation in IDH1 has been found to be an early event during the onset of gliomas [7,8] and thus may exert a vital effect on the initiation of disease.This early onset of IDH1 mutations suggests a more complex role than a mere loss of the enzyme's normal function.Rather than simply losing its enzymatic activity, the IDH1 mutation imparts the enzyme with a neomorphic activity, leading to the reduction of α-ketoglutarate to the oncometabolite R(−)-2-hydroxyglutarate (R-2HG).Reportedly, accumulated 2HG in the brain might increase the risk of developing brain tumors [9][10][11].IDH1 mutation in vivo promote the growth of gliomas and several other malignancies through elevating stem cell number and affecting differentiation [12][13][14][15][16]. IDH1 R132H (mutations occur at a single amino acid residue of IDH1, arginine 132 mutated to histidine) is the most common IDH mutation, present in ∼ 90% of IDH-mutant cases [17].For instance, a highthroughput screen was employed to identify AGI-5198 and MRK-A, selective inhibitors that target IDH1 R132H , which have been discovered to inhibit the generation of the oncometabolite R-2HG and the development of IDH1 R132H -overexpressing gliomas dose-dependently [18,19].Given the association of IDH1 mutations with both an improved prognosis and a pivotal role in disease onset, selective inhibitors targeting IDH1 or R-2HG related signaling pathways are attractive strategies for gliomas treatment regimens [20].
microRNAs (miRNAs) are noncoding oligonucleotides capable of regulating messenger RNA (mRNA) transcript translation and levels in the cytoplasm.One miRNA can regulate several genes as its targets, so miRNA expression profiling may more precisely stratify biologically and clinically relevant subgroups than standard mRNA expression profiling [21,22].Furthermore, the ability to modulate or simulate these oligonucleotides therapeutically via synthetic techniques suggests that these studies may have clinical value [22].Unlike mRNAs, miRNAs are biologically stable and are not susceptible to quick degradation by RNases, facilitating their potentials as prognostic and diagnostic biomarkers, as well as therapeutic targets for gliomas [23][24][25].Numerous miRNAs have been repeatedly found to be dysregulated in many investigations of miRNA expression in gliomas, such as those conducted as part of The Cancer Genome Atlas (TCGA; https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga) and Chinese Glioma Genome Atlas (CGGA; http://www.cgga.org.cn/)projects.miRNAs that are dysregulated during glioma development, aiding or hindering tumorigenesis.
While direct miRNA delivery to the brain has shown promise, a less invasive approach is preferable due to inherent challenges such as reduced resistance to RNase degradation, limited stability, and suboptimal cell uptake of miRNAs [26].Nanoparticles (NPs), especially those fabricated from natural polymers like chitosan (CS), present a promising avenue.CS, a biodegradable and biocompatible polysaccharide consisting of D-glucosamine and N-acetyl-D-glucosamine units connected with b- (1,4) glycosidic linkages [27,28].Its inherent polycationic nature facilitates binding to negatively-charged therapeutics, including miRNAs, ensuring their stability, protection against degradation, and enhanced cellular uptake [27].Given the constraints in miRNA application due to their instability and delivery challenges, the progression of an effective delivery system like CS-NPs shows to be pivotal [26].Chitosan has great potential in the delivery of polynucleotides because of its excellent biological qualities: it is biocompatible, biodegradable, mucoadhesive and non-toxic, bridging the gap between the therapeutic potential of miRNAs and their practical application [29,30].
In this study, differentially expressed miRNAs were analyzed using miRNA expression profiles in IDH1mutant and IDH1-wildtype glioma based on TCGA, CGGA, and online dataset GSE119740; miR-182-5p was selected.IDH1 mutant glioma cells were subjected to treatment with R-2HG and examined for the expression of miR-182-5p and its functions upon R-2HGtreated glioma cells.We analyzed downstream targets of miR-182-5p, investigated the predicted binding of miR-182-5p to target, miR-182-5p regulation of target, and the dynamic effects of the miR-182-5p/target axis on R-2HG-treated glioma cells and xenograft tumor models.CS-NPs(antagomir-182-5p) was synthesized, the characteristics were confirmed, and the anti-tumor effects were investigated in xenograft tumor models in mice.

Clinical sampling
A total of 12 glioma tissue samples (6 with IDH1 mutation and 6 with wild-type IDH1) were obtained from patients receiving surgery at Xiangya Hospital.The glioma IDHl mutation was assessed by postoperative pathological diagnosis of immunohistochemistry and DNA sequencing (Cheerland, Shenzhen, China).All patients involved in the present study did not receive any preoperative radiotherapy or chemotherapy.The pathological stage, grade, and nodal condition were all examined by an experienced pathologist.All experiments were conducted under the approval of the Research Ethics Committee of the Xiangya Hospital.Each patient was consented in a written informed consent form.All the tissues were fixed in formalin or kept at -80 °C before further experiments.

RNA extraction and quantitative real-time PCR (qRT-PCR)
TRIZOL™ (Invitrogen, Carlsbad, USA) was employed to extract total RNA from cells or tissue samples, Prime-Script® Stra Strand Synthesis Kit (TaKaRa, Tokyo, Japan) was utilized to reverse transcribe 2.0 µg of total RNA for synthesis.The quantitative PCR was performed using QuantiTect® SYBR® Green RT-PCR Kit (QIAGEN, Dusseldorf, Germany).mature miR-182-5p, pre-miR-182-5p and CDKN2C expression levels were quantified using the 2 −ΔΔCt method and normalized using U6 or GAPDH as an internal reference.The primer sequences used in qRT-PCR assay are listed in Table S1.

Cell viability by CCK-8
A CCK-8 kit (Sigma-Aldrich, Saint Louis, USA) was applied to assess the viability of target cells.The transfected cells (1 × 10 4 cells/well) were planted on 96-well plates and cultured for 48 h.After that, CCK-8 solution (10 μl/well) was supplemented, and then incubated for 4 h.Using a microplate reader, the absorbance value was obtained at 450 nm.

Cell apoptosis by Flow cytometry
Trypsin digestion (without EDTA) was employed to collect cells.Cell apoptosis was detected using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Vazyme, Nanjing, China) as directed by the protocol of the manufacturer and previous research [34].The data were analyzed using flow cytometry (Novocyte, Agilent, Santa Clara, USA).

Xenograft mouse models
BALB/c nude mice (4 weeks old) were procured from Hunan SLAC laboratory animal company (Changsha, China) for in vivo research.The experiments were carried out in complete accordance with the procedures approved by the Ethics Committee of Xiangya Hospital of Central South University.For subcutaneous xenografts, each nude mouse's right flank was given subcutaneous injection with 5 × 10 6 U251-MG cells (in a total volume of 0.1 ml cell suspension).After tumor formation, the nude mice were then randomized into eight groups (six mice for each).As for in vivo function miR-182-5p and R-2HG, every 7 days, antagomir (5 nmol in 20μl) and/or R-2HG (0.5 µM in 20 µl) was administered via intratumoral injection for 4 weeks.As for the function of antagomirloaded CS-NPs, either antagomir (5 nmol in 20 µl) was administered via intratumoral injection or an equivalent amount of antagomir-loaded CS-NPs was given, and this regimen was continued for a duration of 4 weeks.Tumor volumes were assessed and computed using the following formula: volume (mm 3 ) = length×width 2 /2.After sacrificing the mice, the xenograft tumor cells were separated at the tumor endpoints.Tumor weight was determined.H&E staining was performed to evaluate the pathological alterations in tumor tissue samples.qRT-PCR was conducted to determine miR-182-5p levels within tumor tissue samples.

Hematoxylin and eosin staining (H&E staining)
Mice were anesthetized and sacrificed at the end of the experiment, and tumors were extracted from mice.Tumors were fixed with 4% paraformaldehyde, dehydrated, and embedded with paraffin.Paraffin-embedded blocks were sectioned into 4-µm-thick serial slices, deparaffinized using xylene, and rehydrated using decreasing concentrations of alcohol (100%, 95% and 70%).Slices were washed in dH 2 O, subjected with staining with H&E solution in sequence, and then rinsed in dH 2 O. Next, slices were dehydrated using decreasing concentrations of alcohol and subjected to immersion in xylene prior to mounting in Permount.

Preparation of CS-NPs
CS-NPs were synthesized using complex coacervation method as described by Mao [21].Herein, chitosan (448,869, Sigma-Aldrich) with the following properties was utilized: molecular weight: 50,000-190,000 Da (based on viscosity: 20-300 cP).In short, chitosan was added into 1% acetic acid (0.5 mg/ml), which were stirred at RT for 20-24 h (hour) using a magnetic stirrer, and pH of the solution was adjusted to 6 using NaOH solution.Then, antagomir-182-5p were added into chitosan solution to 50 µg/ml.The chitosan and antagomir-182-5p mixture were stirred for 1 h at RT and ultrasonic for 30 min.The Sodium Tripolyphosphate (TTP) solution (50 mg/ ml) was added dropwise to the chitosan and antagomir-182-5p mixture at N/P ratio of 50 under stirring for 1 h at RT and ultrasonic for 1 h.Lastly, 30-min centrifugation (15,000 rpm/min) was carried out to collect CS-NPs.

Physicochemical characterization of CS-NPs(antagomir-182-5p)
A Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was employed to assess the size and zeta potential of the prepared CS-NPs (antagomir-182-5p).The assessments were carried out three times at pH 7.4 at 25℃.Transmission Electron Microscopy (TEM) was utilized to perform the morphological examination of CS-NPs (antagomir-182-5p).Next, CS-NPs solution was dropped onto a carbon-coated copper grid and dried at RT.A TEM instrument (Hitachi, Tokyo, Japan) was then applied to evaluate the samples.

In vitro release study of antagomir-182-5p from CS-NPs formulation
CS-NPs-antagomir-182-5p were suspended within 1 ml of Tris-EDTA buffer (TE buffer) in RNase free Eppendorf tubes an incubated at 37 °C with shaking at 60 rpm as previous description [37].At appointed time intervals, the supernatant was harvested for evaluation and renewed with new buffer.Quant-iT RiboGreen RNA Assay Kit was employed to determine the quantity of released antagomir-182-5p.

Gel retardation assay
As for antagomir-182-5p integrity, CS-NPs were mixed with loading buffer and loaded on a 2% agarose gel for electrophoresis.CS-NPs and antagomir-182-5p were pretreated with or without RNases and then mixed with loading buffer and loaded on a 2% agarose gel for electrophoresis.The Bio-rad gel documentation system was used to visualize antagomir-182-5p integrity.

Statistical analysis
GraphPad Prism (La Jolla, USA) was applied to analyze the findings of at least three different experiments, and the results were presented in form of mean ± standard deviation (SD).To determine statistical significance, a one-way analysis of variance (ANOVA) and Tukey's multiple comparison test, or Student's t-test were carried out to process data.Correlation analysis was performed according to Pearson's correlation of all data generated in the present study.The significance level was set at P < 0.05.

In vivo functions of miR-182-5p and R-2HG in mice model
As for the in vivo effects of miR-182-5p, xenograft tumor model was established in nude mice and R-2HG and/ or antagomir-182-5p injection was administered as described.Figure 4A-B showed that R-2HG dramatically enlarged tumors, increased tumor weight, and increased tumor volume, while miR-182-5p knockdown remarkably reduced tumors, elevated tumor weight, and boosted tumor volume; when co-transduced, miR-182-5p knockdown partially relieved the tumor-promoting effects of R-2HG.In tumor tissues, the miR-182-5p levels showed to be considerably increased by R-2HG, downregulated by antagomir-182-5p; when co-transduced, the promotive effects of R-2HG on miR-182-5p levels were partially abolished via antagomir-182-5p (Fig. 4C).H&E staining was performed to evaluate the pathological alterations of tumor tissue samples (Fig. 4D).Compared to the model control group, R-2HG elevated mitotic activity and vascular proliferation and promoted cell density in tumor tissues.While there was tumor necrosis and a decrease in cell density and changes in morphology, with irregular cell shapes and dispersed, light purple and unevenly colored nuclei in antagomir-182-5p treatment mice tumor tissues (Fig. 4D).Immunoblotting was conducted to examine CDKN2C, CDK4, Cyclin-D1, p-RB1, RB1, Bax, and Bcl-2 protein contents within tumor tissue samples.Figure 4E shows that R-2 HG remarkably reduced CDKN2C and Bax protein contents, whereas increased Cyclin-D1, CDK4, RB1 phosphorylation and Bcl-2 proteins; miR-182-5p knockdown exerted opposite effects upon these proteins, and partially alleviated the effects of R-2HG.

Discussion
Herein, miR-182-5p expression showed to be increased within IDH1-mutant glioma tissue samples according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples.R-2HG treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p knockdown partially eliminated R-2HG's oncogenic effects upon glioma cells.By direct binding to CDKN2C 3'UTR, miR-182-5p inhibited CDKN2 expression.Regarding cellular functions, CDKN2C knockdown promoted viability, suppressed apoptosis, and relieved cell cycle arrest of R-2HG-treated glioma cells.Furthermore, CDKN2C knockdown partially attenuated the functions of miR-182-5p knockdown upon cell phenotypes.Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition upon cell cycle check point and apoptosis markers.Herein, CS-NPs encapsulated antagomir-182-5p were synthesized and their underlying function as an anti-tumor agent against xenografted tumor in nude mice was explored.We showed that CS-NPs (antagomir-182-5p) had minimal toxicity in mice and exerted a favorable anti-tumor effect on xenograft tumor model in nude mice.
Both enantiomers of 2HG, R-2HG and S-2HG, have been found to be linked to tumor growth through their suppressive roles in αKG (α-ketoglutarate)-dependent dioxygenases.IDH mutations lead to a neomorphic enzymatic activity of the mutated IDH enzymes (mitochondrial IDH2 and cytosolic IDH1), from which the R-2HG is mainly derived, while S-2HG is generated as a result of pathological processes including hypoxia [43].Although the role of R-2HG is complex and seemingly paradoxical, it has commonly been reported as an oncometabolite in gliomas.As aforementioned, AGI-5198 dose-dependently suppressed R-2HG production in mutant enzyme mIDH1.Under near-complete R-2HG inhibition conditions, inhibition of mIDH1 attenuated the proliferation of IDH1-mutated glioma cells without largely affecting genome-wide DNA methylation levels [18].Herein, R-2HG treatment significantly induced malignant behaviors of glioma cells, as manifested as promoted cell miR-182-5p was considered to be an oncogenic miRNA within many malignancies, such as lung carcinoma [44], breast carcinoma [45], prostate carcinoma [46], liver carcinoma [47], colorectal carcinoma [48], and gliomas [49][50][51][52].Reportedly, in glioma cells, miR-182-5p could affect the capacity of cancer cells to proliferate, invade and migrate, as well as the drug-sensitivity.Furthermore, miR-182-5p also enhances glioblastoma angiogenesis.Herein, after inhibiting miR-182-5p by antagomir-182-5p, R-2HG-induced glioma cell viability and R-2HG-suppressed cell apoptosis were partially reversed, confirming its oncogenic role in glioma.Mechanically, miRNAs play their roles via binding to downstream mRNAs [21]; herein, CDKN2C was considered to be a direct downstream target of miR-182-5p.Through direct binding to CDKN2C 3'UTR, miR-182-5p repressed the expression level of CDKN2.Conversely, CDKN2C expression was dramatically decreased in R-2HG-stimulated glioma cells, suggesting that CDKN2C might mediate the effects of miR-182-5p upon R-2HG-induced glioma cells.
Notably, CDKN2C belongs to the INK4/CDKN2 family (CDKN2A [p15], CDKN2B [p16], CDKN2C [p18], and CDKN2D [p19]), which is one of the cyclin-dependent kinase inhibitors which suppress cell cycle development via the crosstalk with CDK4/6 to prevent cyclin D-CDK4/6 complex activation [53].The abnormal CDKN loss resulted in uncontrolled RB phosphorylation and unregulated development via the S phase of the cell cycle, and was found to be associated with the progression of a variety of cancers [54][55][56][57].Consistently, in this study, in addition to enhancing the cell viability and inhibiting cell apoptosis of R-2HG-treated glioma cells, CDKN2C knockdown also relived G1-phase arrest of cell cycle.As for cell cycle check point proteins and apoptotic signaling markers, CDKN2C knockdown increased CDK4, cyclin D1, and Bcl-2 protein contents and promoted RB1 phosphorylation, whereas decreased Bax protein levels.In contrast, miR-182-5p inhibition induced cell cycle arrest, as well as exerted opposite effects on cell cycle and apoptotic signaling markers.More importantly, CDKN2C knockdown partially abolished the tumor-suppressive roles of miR-182-5p inhibition, indicating that miR-182-5p exerts its functions upon R-2HG-treated glioma cells through CDKN2C.In vivo, miR-182-5p inhibition showed significant anti-tumor effects upon xenograft tumor model in nude mice, further confirming the anti-tumor effects of miR-182-5p inhibition.
Considering the excellent delivery properties of CS and NPs [58][59][60], we successfully synthesized CS-NPs(antagomir-182-5p) encapsulating antagomir-182-5p and explored their therapeutic potential against xenografted tumors in nude mice.Morphologically, these nanoparticles exhibited a consistent spherical shape, as observed under TEM, aligning with previous nanoparticle studies [61].Their size, measured at 100 nm, and a polydispersity index (PDI) of 0.25, suggest a uniform distribution, which is crucial for consistent drug delivery [61].The positive zeta potential of + 24 mV indicates stability in suspension, reducing the likelihood of aggregation [62].Notably, the in vitro release profile demonstrated a controlled release of antagomir-182-5p, an essential feature for sustained therapeutic effects.Furthermore, the gel retardation assay and ribonuclease protection assay results collectively highlight the protective role of CS-NPs against RNA degradation, a challenge previously reported in naked RNA-based therapies [63].The transfection efficiency results demonstrated the potential of CS-NPs(antagomir-182-5p) in downregulating miR-182-5p, resulting in an increase in its target protein, CDKN2C.This modulation is consistent with our in vitro results that miR-182-5p inhibition led to the increase in CDKN2C.In vivo, the CS-NPs (antagomir-182-5p) demonstrated safety, as evidenced by the absence of pathological alterations in vital organs and showcased superior anti-tumor effects compared to antagomir-182-5p alone.This potent anti-tumor activity, combined with the observed safety profile, positions CS-NPs (antagomir-182-5p) as a promising candidate for further therapeutic development against tumors.The enhanced anti-tumor effects of CS-NPs(antagomir-182-5p) might stem from the superior controlled-release properties of CS-NPs.
In conclusion, miR-182-5p is elevated while CDKN2C is diminished in IDH1-mutant gliomas and R-2HGtreated glioma cells.This miR-182-5p/CDKN2C dynamic drives the oncogenic effects of R-2HG, impacting cell viability, apoptosis, and the cell cycle.The crafted CS-NPs(antagomir-182-5p) adeptly encapsulate and deliver antagomir-182-5p, amplifying in vivo anti-tumor efficacy and in vivo safety in xenograft tumor model in mouse.Collectively, these insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/CDKN2C axis, offering a promising therapeutic avenue against R-2HG's oncogenic influence in both cellular and mice models.